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Inhibition of S6K1 accounts partially for the anti-inflammatory effects of the arginase inhibitor L-norvaline.

Ming XF, Rajapakse AG, Carvas JM, Ruffieux J, Yang Z - BMC Cardiovasc Disord (2009)

Bottom Line: However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects.Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFalpha-induced activation of NF-kappaB, JNK, p38mapk, while it inhibited p70s6k (S6K1) activity.The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vascular Biology, Department of Medicine, Division of Physiology, University of Fribourg, Switzerland. xiu-fen.ming@unifr.ch

ABSTRACT

Background: Pharmacological inhibition of endothelial arginase-II has been shown to improve endothelial nitric oxide synthase (eNOS) function and reduce atherogenesis in animal models. We investigated whether the endothelial arginase II is involved in inflammatory responses in endothelial cells.

Methods: Human endothelial cells were isolated from umbilical veins and stimulated with TNFalpha (10 ng/ml) for 4 hours. Endothelial expression of the inflammatory molecules i.e. vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin were assessed by immunoblotting.

Results: The induction of the expression of endothelial VCAM-1, ICAM-1 and E-selectin by TNFalpha was concentration-dependently reduced by incubation of the endothelial cells with the arginase inhibitor L-norvaline. However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects. To confirm the role of arginase-II (the prominent isoform expressed in HUVECs) in the inflammatory responses, adenoviral mediated siRNA silencing of arginase-II knocked down the arginase II protein level, but did not inhibit the up-regulation of the adhesion molecules. Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFalpha-induced activation of NF-kappaB, JNK, p38mapk, while it inhibited p70s6k (S6K1) activity. Silencing S6K1 prevented up-regulation of E-selectin, but not that of VCAM-1 or ICAM-1 induced by TNFalpha.

Conclusion: The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells. The anti-inflammatory properties of L-norvaline are partially attributable to its ability to inhibit S6K1.

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Effects of L-norvaline on TNFα-induced activation of various signaling pathways. After pre-treatment of the cells with L-norvaline (Nor. 20 mmol/L), the cells were stimulated with TNFα (10 ng/ml, 15 min) as indicated. The extracts were subjected to immunoblotting to examine the activation of NF-κB by monitoring degradation and phosphorylation of IκB. The activation of JNK, p38mapk and S6K1 pathways were assessed by monitoring phosphorylation of their substrate c-jun, CREB and S6, respectively. Shown are representative blots from four independent experiments.
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Figure 5: Effects of L-norvaline on TNFα-induced activation of various signaling pathways. After pre-treatment of the cells with L-norvaline (Nor. 20 mmol/L), the cells were stimulated with TNFα (10 ng/ml, 15 min) as indicated. The extracts were subjected to immunoblotting to examine the activation of NF-κB by monitoring degradation and phosphorylation of IκB. The activation of JNK, p38mapk and S6K1 pathways were assessed by monitoring phosphorylation of their substrate c-jun, CREB and S6, respectively. Shown are representative blots from four independent experiments.

Mentions: Given the above observations, we then tested whether the anti-inflammatory effects of L-norvaline are attributable to its ability to interfere with signaling pathway(s) involved in expression of the inflammatory molecules. Upon stimulation with TNFα (10 ng/ml, 15 minutes), NF-κB, JNK, p38mapk and S6K1 pathways were activated, as monitored by phosphorylation and degradation of IκB for NF-κB activation, and phosphorylation of the substrate of JNK, p38mapk and S6K1: c-jun, CREB and S6, respectively (Fig. 5, lane 2). Pre-treatment of the cells with L-norvaline (20 mmol/L) did not significantly affect the activation of NF-κB, JNK or p38mapk, but remarkably inhibited S6K1 activation (Fig. 5, lane 3).


Inhibition of S6K1 accounts partially for the anti-inflammatory effects of the arginase inhibitor L-norvaline.

Ming XF, Rajapakse AG, Carvas JM, Ruffieux J, Yang Z - BMC Cardiovasc Disord (2009)

Effects of L-norvaline on TNFα-induced activation of various signaling pathways. After pre-treatment of the cells with L-norvaline (Nor. 20 mmol/L), the cells were stimulated with TNFα (10 ng/ml, 15 min) as indicated. The extracts were subjected to immunoblotting to examine the activation of NF-κB by monitoring degradation and phosphorylation of IκB. The activation of JNK, p38mapk and S6K1 pathways were assessed by monitoring phosphorylation of their substrate c-jun, CREB and S6, respectively. Shown are representative blots from four independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664787&req=5

Figure 5: Effects of L-norvaline on TNFα-induced activation of various signaling pathways. After pre-treatment of the cells with L-norvaline (Nor. 20 mmol/L), the cells were stimulated with TNFα (10 ng/ml, 15 min) as indicated. The extracts were subjected to immunoblotting to examine the activation of NF-κB by monitoring degradation and phosphorylation of IκB. The activation of JNK, p38mapk and S6K1 pathways were assessed by monitoring phosphorylation of their substrate c-jun, CREB and S6, respectively. Shown are representative blots from four independent experiments.
Mentions: Given the above observations, we then tested whether the anti-inflammatory effects of L-norvaline are attributable to its ability to interfere with signaling pathway(s) involved in expression of the inflammatory molecules. Upon stimulation with TNFα (10 ng/ml, 15 minutes), NF-κB, JNK, p38mapk and S6K1 pathways were activated, as monitored by phosphorylation and degradation of IκB for NF-κB activation, and phosphorylation of the substrate of JNK, p38mapk and S6K1: c-jun, CREB and S6, respectively (Fig. 5, lane 2). Pre-treatment of the cells with L-norvaline (20 mmol/L) did not significantly affect the activation of NF-κB, JNK or p38mapk, but remarkably inhibited S6K1 activation (Fig. 5, lane 3).

Bottom Line: However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects.Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFalpha-induced activation of NF-kappaB, JNK, p38mapk, while it inhibited p70s6k (S6K1) activity.The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vascular Biology, Department of Medicine, Division of Physiology, University of Fribourg, Switzerland. xiu-fen.ming@unifr.ch

ABSTRACT

Background: Pharmacological inhibition of endothelial arginase-II has been shown to improve endothelial nitric oxide synthase (eNOS) function and reduce atherogenesis in animal models. We investigated whether the endothelial arginase II is involved in inflammatory responses in endothelial cells.

Methods: Human endothelial cells were isolated from umbilical veins and stimulated with TNFalpha (10 ng/ml) for 4 hours. Endothelial expression of the inflammatory molecules i.e. vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin were assessed by immunoblotting.

Results: The induction of the expression of endothelial VCAM-1, ICAM-1 and E-selectin by TNFalpha was concentration-dependently reduced by incubation of the endothelial cells with the arginase inhibitor L-norvaline. However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects. To confirm the role of arginase-II (the prominent isoform expressed in HUVECs) in the inflammatory responses, adenoviral mediated siRNA silencing of arginase-II knocked down the arginase II protein level, but did not inhibit the up-regulation of the adhesion molecules. Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFalpha-induced activation of NF-kappaB, JNK, p38mapk, while it inhibited p70s6k (S6K1) activity. Silencing S6K1 prevented up-regulation of E-selectin, but not that of VCAM-1 or ICAM-1 induced by TNFalpha.

Conclusion: The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells. The anti-inflammatory properties of L-norvaline are partially attributable to its ability to inhibit S6K1.

Show MeSH
Related in: MedlinePlus